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PubMed1997

Ligation-mediated PCR amplification of specific fragments from a class-II restriction endonuclease total digest.

A method is described which permits the ligation- mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest. Feasibility was tested using Bcl I and phage lambda DNA as a model enzyme and amplicon system, respectively. Bcl I is one of many widely used restriction enzymes which cleave at palindromic recognition sequences and leave 5'-protruding ends of defined sequence. Using a single pair of universal primers, a given fragment can be specifically amplified after joining the fragments to adaptors consisting of a duplex primer region and a 9-nucleotide protruding single-stranded 5'-end containing the sequence complementary to the cleaved restriction site and a 4-nucleotide 'indexing sequence.' The protruding strand anneals to a restriction fragment by displacing its corresponding strand in the same fragment-specific indexing sequence located juxtaposed to the restriction site. The adaptor is covalently linked to the restriction fragment by T4 DNA ligase, and amplification is carried out under conditions for long-distance PCR using the M13 forward and reverse primers. The technique discriminated robustly between mismatches and perfect matches for the 16 indexing sequences tested to allow individual lambda Bcl I fragments to be amplified from their respective adaptor pairs. A strategy is proposed enabling a non-cloning approach to the accession, physical mapping and sequencing of genomic DNA. The method could also have application in high-throughput genetic mapping and fingerprinting and should expand the enzyme base for ligation- mediated indexing technology which has previously been limited to the Class-IIS and IP restriction endonucleases.

Bacteriophage lambda↗
PubMed1998

Identification of fragments of human transcripts froma defined chromosomal region: representational difference analysis of somatic cell hybrids.

We have tested representational difference analysis of cDNAs from somatic cell hybrids as a means to directly isolate expressed sequences derived from a defined chromosomal region. To this end, the hamster-human somatic cell hybrid Q1Z, which carries Xq28 as the only human chromosomal fragment, was used as Tester and the parental hamster cell line Y21 as Driver. After two rounds of subtraction, two major products of 510 and 307 bp were obtained, derived from the highly expressed human Xq28-derived QM gene and from a hamster repeat sequence strongly up-regulated in Q1Z, respectively. In a second subtraction experiment these fragments were added to the driver, to prevent their reappearance. After three rounds of subtraction a more complex difference product was obtained. Of 26 different fragments analysed, 12 fragments were derived from Xq28-derived genes, 10 of which were from known genes. One fragment was derived from a hamster gene strongly up-regulated in Q1Z. These results demonstrate that cDNA RDA can be used to isolate gene fragments from defined chromosomal regions and that suppression with major products, derived from highly expressed genes, is advantageous to isolate larger number of fragments, presumably derived from rarer transcripts.

Animals↗
PubMed1998

Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is one of the most powerful methods for mutation detection currently available. For successful application the appropriate selection of PCR fragments and PCR primers is crucial. The sequence of interest should always be within the domain with the lowest melting temperature. When more than one melting domain is present the fragment is generally divided into several smaller ones. This, however, is not always necessary. We found that simple modifications of PCR fragments and primer sequences may substantially reduce the number of amplicons required. Furthermore, by plotting the (natural) melting curves of fragments without a GC-clamp, we could explain why fragments theoretically perfect for DGGE in practice failed to reveal mutations. Alternative fragment selection and the use of modified primers (addition of T/A or G/C tails) result in the detection of mutations that originally remained undetected. Our studies extend the utility of DGGE by using a minimum of PCR fragments and achieving a maximum of mutation detection.

Base Composition↗
PubMed2002

Commonly conserved genetic fragments revealed by genome profiling can serve as tracers of evolution.

We developed a method to produce, identify and analyze DNA fragments for the purpose of taxonomic classification. Genome profiling (GP) is a strategy that identifies genomic DNA fragments common to closely related species without prior knowledge of the DNA sequence. Random PCR, one of the key technologies of GP, is used to produce fragments and may be used even when there are mutations at the priming site. These fragments can then be distinguished based on the information of mobility and melting pattern when subjected to temperature gradient gel electrophoresis (TGGE). Corresponding fragments among several species, designated as commonly conserved genetic fragments (CCGFs), likely have the same genetic origin or correspond to the same gene. The criteria for identification of CCGFs has been defined and presented here. To assess this prediction, some of the fragments were sequenced and were confirmed to be CCGFs. We show that genome profiles bearing evolutionarily conserved CCGFs can be used to classify organisms and trace evolutionary pathways, among other profound applications.

Bacillus↗
PubMed1978

The ovalbumin split gene: molecular cloning of Eco RI fragments "c" and "d".

The Eco RI fragments "c" and "d" of the ovalbumin gene (1, 2) have been isolated by molecular cloning. Restriction enzyme mapping and electron microscopy have confirmed that the two fragments contain the same ovalbumin mRNA coding sequences. These sequences are split into two regions which have been mapped in fragments "c" and "d". There is no evidence that the ovalbumin mRNA sequences contained in these fragments could be further interrupted. Our results confirm that the presence of Eco RI fragment "d" in some chickens is due to the existence of an allelic variant of the ovalbumin gene which contains an additional Eco RI site within the region corresponding to Eco RI fragment "c". This additional Eco RI site appears to be the main difference between the two alleles. Finally, our results provide a direct demonstration that most of the ovalbumin mRNA sequences are encoded for by Eco RI fragments "a", "b" and "c".

Coliphages↗
PubMed1979

Propagation of restriction fragments from the mitochondrial DNA of Saccharomyces cerevisiae in E. coli by means of plasmid vectors.

Some of the EcoRI fragments of yeast (Saccharomyces cerevisiae) mitochondrial DNA were cloned into E. coli using plasmid pMB9. The five smallest fragments in molecular weight appeared to be preferentially retained by E coli; partial fragments derived from larger mitochondrial DNA fragments were also found. One of the fragments, R7 (2.4 kb), may contain the OII gene. Cloned R7 DNA was stable under a variety of growth conditions, but showed some changes in molecular weight after transfer to different E. coli strains. Fragment R7 is transcribed in minicells, producing RNA that hybridizes specifically to mitochondrial DNA. Both DNA strands are transcribed, in contrast to the asymmetric transcription found in mitochondria. No new polypeptides were observed in minicells containing cloned fragment 7.

DNA Restriction Enzymes↗
PubMed2005

Conversion of nucleotide sequence with single-stranded DNA fragment prepared from phagemid DNA.

The correction of a mutated gene is a highly attractive approach for gene therapy. This in vivo mutagenesis method will also be an effective tool in biotechnology. However, the current small fragment homologous replacement (SFHR) method with a heat-denatured double-stranded PCR fragment yielded the low correction efficiency. Single-stranded DNA fragments were prepared from single-stranded phagemid DNAs and tested in a gene correction assay with a Hyg-EGFP fusion gene inactivated by a substitution mutation, as a model target. A 606-nt sense, single-stranded DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. On the other hand, correction of frameshift mutations with the sense single-stranded DNA fragment were 2-3-fold as efficient as that with the PCR fragment. These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes.

Bacteriophages↗
PubMed1987

Conformation of the constant fragment of the immunoglobulin light chain: effect of cleavage of the polypeptide chain and the disulfide bond.

In order to understand the conformations and stabilities of the immunoglobulin domains, a derivative of the type-lambda constant fragment in which the peptide bond Arg190-Ser191 is cleaved and a derivative of the type-kappa constant fragment in which the peptide bond Arg142-Glu143 is cleaved were prepared by limited proteolysis with the proteinase from mouse submaxillary gland, endoproteinase Arg-C [EC 3.4.21.40]. The cleaved peptide bond of each derivative is located in the loop formed by the intrachain disulfide bond and the two peptides formed are linked by the disulfide bond. The two nicked CL fragments did not assume any ordered conformation. On the other hand, a derivative in which the intrachain disulfide bond is reduced had a conformation very similar to that of the intact CL fragment, although the stability was considerably low. Since the entropy of the nicked fragment should be nearly the same as that of the reduced CL fragment in the unfolded state, a destabilizing effect of cleavage of the polypeptide bond in the folded state in addition to the entropic effect in the unfolded state seems necessary to account for the conformations of the nicked CL fragments.

Ammonium Sulfate↗
PubMed1987

Fluorescence of the tryptophyl residues of the constant fragment of the immunoglobulin light chain.

The constant (CL) domain of the type-lambda immunoglobulin light chain contains two tryptophyl residues. The fluorescence lifetimes of the CL fragment and its two modified forms, the reduced CL, and reduced and alkylated CL fragments, were measured at pH 7.5 and 25 degrees C. The fluorescence decay kinetics of these fragments were described in terms of two lifetimes. The static and dynamic quenching reactions by acrylamide of the tryptophyl residues of these fragments were measured and their dynamic properties were compared with the static properties reported previously (Goto, Y. & Hamaguchi, K. (1979) J. Biochem. 86, 1433-1441; Goto, Y. & Hamaguchi, K. (1986) Biochemistry 25, 2821-2828; Ashikari, Y., Arata, Y., & Hamaguchi, K. (1985) J. Biochem. 97, 517-528). It was found that although the static conformation of the reduced CL fragment is very similar to that of the intact CL fragment, the dynamic conformations of these two fragments differ considerably.

Acrylamides↗
PubMed1997

Fragmented and scrambled mitochondrial ribosomal RNA coding regions among green algae: a model for their origin and evolution.

Mitochondrial ribosomal RNA coding regions in the only three green algal taxa investigated to date are fundamentally different in that they are continuous in Prototheca wickerhamii, but highly fragmented and scrambled in Chlamydomonas reinhardtii and Chlamydomonas eugametos. To gain more insight into the mode of evolution of fragmented and scrambled mitochondrial ribosomal RNA (rRNA) genes within the green algal group, this work (1) provides additional information on fragmentation patterns of mitochondrial small- and large-subunit (SSU and LSU) rRNAs that strongly supports the concept of a gradual increase in the extent of discontinuity of mitochondrial rRNAs among chlorophycean green algae and (2) reports the first example of fragmented and scrambled mitochondrial LSU rRNA coding regions in a green algal taxon outside the Chlamydomonas group. The present study (1) suggests that the scrambling of the mitochondrial rRNA coding regions may have occurred early in the evolution of fragmented and scrambled mitochondrial rRNA genes within the chlorophycean green algal group, most likely in parallel with the fragmentation events, (2) proposes recombination as a possible mechanism involved in the evolution of these mitochondrial rRNA genes, and (3) presents a hypothetical pathway for converting continuous mitochondrial rRNA genes into the highly fragmented and scrambled rRNA coding regions of Chlamydomonas through a series of recombinatorial events between short repeated sequences.

Animals↗
PubMed1996

Proteoglycan-degrading activity associated with the 40 kDa collagen-binding fragment of fibronectin.

The osteoarthritis (OA) process is characterized by the progressive destruction of articular cartilage. There is a loss of cartilage proteoglycan content and disorganization of the collagen network, as well as an increase in other non-collagenous protein such as fibronectin (Fn). Increased proteolytic activity may lead to the degradation of native Fn and generation of Fn proteolytic fragments. Among them, the 45 kDa collagen-binding Fn fragment can be autoactivated in vitro into a 40 kDa fragment. This 40 kDa fragment induces an average of 30% of proteoglycan release per day from human OA cartilage explants and can degrade proteoglycan using dead cartilage sections. Proteoglycan-degrading activity related to the 40 kDa Fn fragment was decreased up to 66% by fetal calf serum (10%), but was not prevented by protein synthesis inhibitors (cycloheximide or actinomycin D). The action of this 40 kDa Fn fragment was greater on OA than on normal cartilage. This study suggests that enzymatic activity induced by the 40 kDa collagen-binding fragment of Fn might be involved in cartilage matrix turnover.

Cartilage↗
PubMed2004

Functional characterization of the 5' flanking region of the BACE gene: identification of a 91 bp fragment involved in basal level of BACE promoter expression.

Pathological characteristics of Alzheimer's disease (AD) include amyloid-beta (Abeta) plaques. Abeta is derived from the Abeta peptide precursor protein (APP) by gamma- and beta-secretases, the latter known as beta-site APP-cleaving enzyme 1 (BACE1, or herein BACE). We have also described potentially important regions in the promoter of BACE, which may regulate its activity (1). Herein, we have functionally dissected the regulatory regions within the BACE promoter into areas containing positive and negative regulatory elements. The 4.1 kb promoter region (-3765/+364, +1 being the transcription start site [TSS]) includes positive regulatory element in the -2975 to -2062 region flanked on either side by negative regulatory elements at -3764/-2975 and -2062/-1056. This is separated from the minimal promoter and 5' UTR by a neutral region of roughly 700 base pairs (bp). A 91 bp fragment (224/314) is the shortest region with a significant reporter gene activity and constitutes the minimal promoter element for BACE. Neuronal preference of the promoter was apparent in the 141 bp fragment (224/364) that contained the 91 bp fragment. Gel shift results also suggest the strongest signal of DNA-protein interaction with the 91 bp fragment in neuronal nuclear extracts. This interaction was strongly blocked by an activator protein (AP)2 binding oligomer. Super gel shift assays suggest that both the AP2-binding oligomer and the 91 bp fragment interfered with each other's binding capacity. An AP2 sequence is predicted to occur within the 91 bp fragment. We also found stimulating protein (SP)1-binding sites in this region of the promoter. Thus, functional and gel shift analysis indicate that the 91 bp fragment containing the AP2 and SP1 binding sites constitutes the core promoter region of BACE. Changes in the activity of this region could play an important role in regulating BACE activity in neurons.

5' Flanking Region↗
PubMed1996

DNA fragmentation and HSP70 protein induction in hippocampus and cortex occurs in separate neurons following permanent middle cerebral artery occlusions.

DNA nick end-labeling (TUNEL) and heat shock protein (HSP)70 immunocytochemistry were performed on the same brain sections 1 (n = 6), 3 (n = 12), and 7 (n = 7) days following permanent middle cerebral artery (MCA) occlusions produced in adult rats using the endovascular carotid suture method. In the cortex at 1 and 3 days following MCA occlusions, HSP70 immunoreactive neurons were located outside areas of infarction and showed little evidence of DNA fragmentation. HSP70-stained cortical neurons were intermingled with TUNEL cells near the infarct, but extended for greater distances away from the infarct. DNA fragmentation occurred in CA1 hippocampal neurons in 39% of the animals at 1 and 3 days following ipsilateral MCA occlusion. Bilateral DNA fragmentation occurred in CA1 neurons in one subject. HSP70 protein was expressed in CA1 hippocampal neurons in nine of 18 (50%) animals at 1 and 3 days following MCA occlusions, including all animals that exhibited hippocampal DNA fragmentation. Three animals had bilateral expression of HSP70 in CA1 neurons. Cells that stained for either HSP70 protein or DNA fragmentation existed in close proximity to one another. Approximately 5-7% of HSP70-stained cells were TUNEL stained and 3% of TUNEL-positive cells also stained for HSP70. There was no HSP70 staining or DNA fragmentation in the brains of sham-operated controls (n = 4) or in the brains of animals 7 days following MCA occlusions. These data suggest that ischemic cells capable of translating HSP70 protein generally do not undergo DNA fragmentation. These data support the concept that most HSP70 protein-containing neurons in the cortical "penumbra" and hippocampus survive ischemic injury and are "reversibly injured." It is shown that CA1 hippocampal pyramidal neurons die or are reversibly injured in approximately 50% of animals following permanent MCA occlusions. Although the mechanism of this hippocampal injury is unknown, it could relate to transynaptic activation of N-methyl-D-aspartate (NMDA) receptors that mediate induction of early genes in hippocampus.

Animals↗
PubMed2000

Intraoperative localization of retained metallic fragments in missile wounds.

OBJECTIVE: Patients with missile wounds often retain fragments of different materials within their bodies. They are usually of no clinical consequence, but sometimes they can cause complications. Furthermore, the presence of metallic objects is a contraindication for magnetic resonance imaging. Therefore, the fragments should be removed whenever possible. A new metal detector designed for intraoperative use and a method that allows exact localization and removal of conductive fragments is presented. METHODS: Practical experience shows that the volume of missile fragments varies by a factor of 1,000. Different materials can be detected and located at distances usually not greater than 50 mm. An adaptive electronic amplifier was used in this instrument to locate missile fragments of different sizes and magnetic properties at different distances. Environmental disturbances such as electrical wires and external magnetic fields, and the limited dynamics of human perception, were compensated for by the spatial selectivity and adaptive sensitivity of the instrument. The source of the alternating magnetic field was placed in a hand-held probe connected to the instrument, with which small changes in the magnetic field were detected and exploited for the location of metallic fragments. RESULTS: The boundaries of detectability and localizability by the device have been determined for different missile fragments to demonstrate the diagnostic feasibility and limitations of the method. CONCLUSION: The successful use of the method, in cases where other imaging procedures are either not available or ineffective, is demonstrated by two cases of patients with gunshot wounds.

Adult↗
PubMed1998

Posterior epidural migration of an extruded lumbar disc fragment causing cauda equina syndrome. Clinical and magnetic resonance imaging evaluation.

STUDY DESIGN: Retrospective case report. OBJECTIVES: To illustrate the rare occurrence of cauda equina syndrome resulting from posterior epidural disc fragment migration documented by magnetic resonance imaging. SUMMARY OF BACKGROUND DATA: Disc fragment migration patterns are generally limited by the attachments of the posterior longitudinal ligament and its associated "midline septum" and "peridural" or "lateral membrane." The majority of symptomatic lumbar disc herniations are noted to be in a lateral position with resultant nerve root irritation. Although caudal, rostral, and lateral migration of disc fragments are common, posterior epidural migration of an extruded disc fragment has been reported only rarely, and only once in association with cauda equina syndrome. METHODS: The patient described in this report presented with an acute cauda equina syndrome. Investigation with magnetic resonance imaging revealed a posterior epidural mass, which was hypointense on T1-weighted and T2-weighted images. Postgadolinium imaging showed rim enhancement of the lesion. The preoperative differential diagnosis included epidural abscess, hematoma, or neoplasm. An urgent decompressive lumbar laminectomy was performed. At surgery, the lesion proved to be a massive extruded disc fragment. RESULTS: At the 1-year follow-up examination, the patient had recovered full motor, sensory, urologic, and sexual function. CONCLUSIONS: Extruded disc fragments may migrate posterior to the thecal sac. Magnetic resonance images of a posterior disc fragment may mimic those of other more common posterior epidural lesions.

Cauda Equina↗
PubMed2005

How significant are clinically insignificant residual fragments following lithotripsy?

PURPOSE OF REVIEW: Extracorporeal shock-wave lithotripsy had revolutionized the treatment of kidney stones. Residual fragments smaller than 4 or 5 mm which are asymptomatic and likely to pass spontaneously are termed as clinically insignificant residual fragments. This article reviews the current literature with regards to the management of clinically insignificant residual fragments. RECENT FINDINGS: However, the term is controversial because no consensus has been reached regarding its exact definition or interval after extracorporeal shock-wave lithotripsy to evaluate its presence. While most small residual stone fragments might be managed expectantly, a significant number of patients might become symptomatic or require intervention. These so-called clinically insignificant residual fragments have the potential to cause obstruction and are important risk factors for stone recurrence and re-growth. SUMMARY: Residual fragments require close monitoring for stone growth, potential complications and subsequent intervention. Secondary procedures may be applied selectively to those patients who have significant symptoms of obstruction associated with the residual stone. Medical therapy might play an important role in the management of residual fragments.

Humans↗
PubMed1982

Cloning of DNA fragments from the left end of the adenovirus type 12 genome: transformation by cloned early region 1.

The human adenovirus serotype 12 (Ad-12) EcoRI-C DNA fragment (0 to 16.5 map units) was cloned in the plasmid vector pAT153. This cloned Ad-12 EcoRI-C DNA fragment was subcloned generating recombinant plasmids which contained the Ad-12 SalI-C fragment (0 to 10.3 map units), the Ad-12 HindIII-G fragment (0 to 6 X 8 map units) and the Ad-12 AccI-H fragment (0 to 4 X 7 map units). Thus, we constructed recombinant plasmids which contain Ad-12 DNA sequences which represent all or part of the virus transforming gene region. The capacity of the cloned Ad-12 EcoRI-C DNA fragment to transform rat cells in vitro was assessed using the focus assay on primary cultures of rat cells. The specific transforming activity of this recombinant plasmid was in the same range as that found for intact Ad-12 DNA. Transformed foci which were induced by the cloned Ad-12 EcoRI-C DNA fragment were established as cell lines and the presence of Ad-12 DNA in these lines was demonstrated using the Southern blotting technique.

Adenoviruses, Human↗
PubMed2000

Quantum tunneling fragmentation model

A nonthermal quantum mechanical statistical fragmentation model based on tunneling of particles through potential barriers is studied in compact two- and three-dimensional systems. It is shown that this fragmentation dynamics gives origin to several static and dynamic scaling relations. The critical exponents are found and compared with those obtained in classical statistical models of fragmentation of general interest, in particular with thermal fragmentation involving classical processes over potential barriers. Besides its general theoretical interest, the fragmentation dynamics discussed here is complementary to classical fragmentation dynamics of interest in chemical kinetics and can be useful in the study of a number of other dynamic processes such as nuclear fragmentation.

Journal Article↗